Rab26 restricts insulin secretion via sequestering Synaptotagmin-1.

Rab26 is known to regulate multiple membrane trafficking events, but its role in insulin secretion in pancreatic ß cells remains unclear despite it was first identified in the pancreas. In this study, we generated Rab26-/- mice through CRISPR/Cas9 technique. Surprisingly, insulin levels in the blood of the Rab26-/- mice do not decrease upon glucose stimulation but conversely increase. Deficiency of Rab26 promotes insulin secretion, which was independently verified by Rab26 knockdown in pancreatic insulinoma cells. Conversely, overexpression of Rab26 suppresses insulin secretion in both insulinoma cell lines and isolated mouse islets. Islets overexpressing Rab26, upon transplantation, also failed to restore glucose homeostasis in type 1 diabetic mice. Immunofluorescence microscopy revealed that overexpression of Rab26 results in clustering of insulin granules. GST-pulldown experiments reveal that Rab26 interacts with synaptotagmin-1 (Syt1) through directly binding to its C2A domain, which interfering with the interaction between Syt1 and SNAP25, and consequently inhibiting the exocytosis of newcomer insulin granules revealed by TIRF microscopy. Our results suggest that Rab26 serves as a negative regulator of insulin secretion, via suppressing insulin granule fusion with plasma membrane through sequestering Syt1.

Dynein Is Required for Rab7-Dependent Endosome Maturation, Retrograde Dendritic Transport, and Degradation.

In all cell types, endocytosed cargo is transported along a set of endosomal compartments, which are linked maturationally from early endosomes (EEs) via late endosomes (LEs) to lysosomes. Lysosomes are critical for degradation of proteins that enter through endocytic as well as autophagic pathways. Rab7 is the master regulator of early-to-late endosome maturation, motility, and fusion with lysosomes. We previously showed that most degradative lysosomes are localized in the soma and in the first 25 µm of the dendrite and that bulk degradation of dendritic membrane proteins occurs in/near the soma. Dendritic late endosomes therefore move retrogradely in a Rab7-dependent manner for fusion with somatic lysosomes. We now used cultured E18 rat hippocampal neurons of both sexes to determine which microtubule motor is responsible for degradative flux of late endosomes. Based on multiple approaches (inhibiting dynein/dynactin itself or inhibiting dynein recruitment to endosomes by expressing the C-terminus of the Rab7 effector, RILP), we now demonstrate that net retrograde flux of late endosomes in dendrites is supported by dynein. Inhibition of dynein also delays maturation of somatic endosomes, as evidenced by excessive accumulation of Rab7. In addition, degradation of dendritic cargos is inhibited. Our results also suggest that GDP-GTP cycling of Rab7 appears necessary not only for endosomal maturation but also for fusion with lysosomes subsequent to arrival in the soma. In conclusion, Rab7-dependent dynein/dynactin recruitment to dendritic endosomes plays multifaceted roles in dendritic endosome maturation as well as retrograde transport of late endosomes to sustain normal degradative flux.SIGNIFICANCE STATEMENT Lysosomes are critical for degradation of membrane and extracellular proteins that enter through endocytosis. Lysosomes are also the endpoint of autophagy and thus responsible for protein and organelle homeostasis. Endosomal-lysosomal dysfunction is linked to neurodegeneration and aging. We identify roles in dendrites for two proteins with links to human diseases, Rab7 and dynein. Our previous work identified a process that requires directional retrograde transport in dendrites, namely, efficient degradation of short-lived membrane proteins. Based on multiple approaches, we demonstrate that Rab7-dependent recruitment of dynein motors supports net retrograde transport to lysosomes and is needed for endosome maturation. Our data also suggest that GDP-GTP cycling of Rab7 is required for fusion with lysosomes and degradation, subsequent to arrival in the soma.

SNARE proteins in membrane trafficking.

SNAREs are the core machinery mediating membrane fusion. In this review, we provide an update on the recent progress on SNAREs regulating membrane fusion events, especially the more detailed fusion processes dissected by well-developed biophysical methods and in vitro single molecule analysis approaches. We also briefly summarize the relevant research from Chinese laboratories and highlight the significant contributions on our understanding of SNARE-mediated membrane trafficking from scientists in China.

RILP Induces Cholesterol Accumulation in Lysosomes by Inhibiting Endoplasmic Reticulum-Endolysosome Interactions.

Endoplasmic reticulum (ER)-endolysosome interactions regulate cholesterol exchange between the ER and the endolysosome. ER-endolysosome membrane contact sites mediate the ER-endolysosome interaction. VAP-ORP1L (vesicle-associated membrane protein-associated protein- OSBP-related protein 1L) interaction forms the major contact site between the ER and the lysosome, which is regulated by Rab7. RILP (Rab7-interacting lysosomal protein) is the downstream effector of Rab7, but its role in the organelle interaction between the ER and the lysosome is not clear. In this study, we found RILP interacts with ORP1L to competitively inhibit the formation of the VAP-ORP1L contact site. Immunofluorescence microscopy revealed that RILP induces late endosome/lysosome clustering, which reduces the contact of endolysosomes with the ER, interfering with the ER-endolysosome interaction. Further examination demonstrated that over-expression of RILP results in the accumulation of cholesterol in the clustered endolysosomes, which triggers cellular autophagy depending on RILP. Our results suggest that RILP interferes with the ER-endolysosome interaction to inhibit cholesterol flow from the endolysosome to the ER, which feedbacks to trigger autophagy.

SNAP23 decreases insulin secretion by competitively inhibiting the interaction between SNAP25 and STX1A.

SNAP25 is a core protein of the SNARE complex, which mediates stimulus-dependent secretion of insulin from the pancreatic ß cells. SNAP23 is a SNAP25 homolog, however, the functional role of SNAP23 in the exocytic secretion of insulin is not known. Therefore, in the present study, we investigated the functional role of SNAP23 in the insulin secretory pathway. Our results demonstrated that over-expression of SNAP23 inhibited the secretion of insulin from the INS-1 cells. Conversely, SNAP23 depletion increased insulin secretion. Mechanistically, overexpression of SNAP23 decreased SNARE complex formation by blocking the binding of SNAP25 to STX1A. The full-length SNAP23 protein with the N-terminal and C-terminal SNARE binding domains was required for competition. Moreover, SNAP23 serine 95 phosphorylation plays a crucial function in insulin secretion by enhancing the interaction between SNAP23 and STX1A. The present study presents a new pathway regulating insulin secretion. Therefore, SNAP23 may be a potential therapeutic target for diabetes mellitus.

The immunity-related GTPase IRGC mediates interaction between lipid droplets and mitochondria to facilitate sperm motility.

The immunity-related GTPases (IRGs) belong to the interferon-inducible GTPase protein family, which mediates cell autonomous and innate immunity response to intracellular pathogens. Yet, the cellular and physiological function of IRGC, a member of the IRG subfamily, has not been elucidated. Here, we show that testis-specific IRGC is specifically and highly expressed in mature spermatozoa and is required for sperm motility. IRGC induction results in the clustering of lipid droplets and initiation of their physical contact with mitochondria. When examining clinical semen samples, IRGC expression is significantly lower in asthenozoospermia patients relative to healthy individuals. These unique effects of IRGC identify it as an important player in sperm motility, and show the potential of lipid metabolism-targeting therapeutic intervention aimed at controlling asthenozoospermia.

The UDPase ENTPD5 regulates ER stress-associated renal injury by mediating protein N-glycosylation.

Impaired protein N-glycosylation leads to the endoplasmic reticulum (ER) stress, which triggers adaptive survival or maladaptive apoptosis in renal tubules in diabetic kidney disease (DKD). Therapeutic strategies targeting ER stress are promising for the treatment of DKD. Here, we report a previously unappreciated role played by ENTPD5 in alleviating renal injury by mediating ER stress. We found that ENTPD5 was highly expressed in normal renal tubules; however, ENTPD5 was dynamically expressed in the kidney and closely related to pathological DKD progression in both human patients and mouse models. Overexpression of ENTPD5 relieved ER stress in renal tubular cells, leading to compensatory cell proliferation that resulted in hypertrophy, while ENTPD5 knockdown aggravated ER stress to induce cell apoptosis, leading to renal tubular atrophy and interstitial fibrosis. Mechanistically, ENTPD5-regulated N-glycosylation of proteins in the ER to promote cell proliferation in the early stage of DKD, and continuous hyperglycemia activated the hexosamine biosynthesis pathway (HBP) to increase the level of UDP-GlcNAc, which driving a feedback mechanism that inhibited transcription factor SP1 activity to downregulate ENTPD5 expression in the late stage of DKD. This study was the first to demonstrate that ENTPD5 regulated renal tubule cell numbers through adaptive proliferation or apoptosis in the kidney by modulating the protein N-glycosylation rate in the ER, suggesting that ENTPD5 drives cell fate in response to metabolic stress and is a potential therapeutic target for renal diseases.

Metrnl Alleviates Lipid Accumulation by Modulating Mitochondrial Homeostasis in Diabetic Nephropathy.

Ectopic lipid accumulation in renal tubules is closely related to the pathogenesis of diabetic kidney disease (DKD), and mitochondrial dysfunction is thought to play a key role in lipid accumulation. Therefore, maintaining mitochondrial homeostasis holds considerable promise as a therapeutic strategy for the treatment of DKD. Here, we report that the Meteorin-like (Metrnl) gene product mediates lipid accumulation in the kidney and has therapeutic potential for DKD. We confirmed the reduced expression of Metrnl in renal tubules, which was inversely correlated with DKD pathological changes in human patients and mouse models. Functionally, pharmacological administration of recombinant Metrnl (rMetrnl) or Metrnl overexpression could alleviate lipid accumulation and inhibit kidney failure. In vitro, rMetrnl or Metrnl overexpression attenuated palmitic acid-induced mitochondrial dysfunction and lipid accumulation in renal tubules accompanied by maintained mitochondrial homeostasis and enhanced lipid consumption. Conversely, shRNA-mediated Metrnl knockdown diminished the protective effect on the kidney. Mechanistically, these beneficial effects of Metrnl were mediated by the Sirt3-AMPK signaling axis to maintain mitochondrial homeostasis and through Sirt3-uncoupling protein-1 to promote thermogenesis, consequently alleviating lipid accumulation. In conclusion, our study demonstrates that Metrnl regulated lipid metabolism in the kidney by modulating mitochondrial function and is a stress-responsive regulator of kidney pathophysiology, which sheds light on novel strategies for treating DKD and associated kidney diseases. ARTICLE HIGHLIGHTS: Metrnl is expressed in renal tubules and is reduced under diabetic conditions. The concentration of Metrnl in the kidney is correlated with lipid accumulation and serum creatinine. Metrnl-specific overexpression in the kidney or recombinant Metrnl administration alleviates renal injuries in diabetic mice. Metrnl regulates renal tubules lipid metabolism through Sirt3-AMPK/UCP1 signaling axis-mediated mitochondrial homeostasis.

The emerging role of VHS domain-containing Tom1, Tom1L1 and Tom1L2 in membrane trafficking.

The maintenance of cellular homeostasis and execution of regulatory mechanisms to dynamically govern various cellular processes require the correct delivery of proteins to their target subcellular compartments. It is estimated that over 30% of the proteins encoded by the human genome, projected to encode about 25 000 proteins and other macromolecules, are delivered to the secretory and endocytic pathways where movement of proteins between various compartments is primarily mediated by vesicles/carriers budding from one compartment for delivery to another. Sorting of cargo proteins into budding vesicles/carriers is mediated by adaptors that link the cargo proteins to the coat proteins. The adaptor function of VHS domain proteins, GGA proteins, STAM proteins and Hrs is well-established and is evolutionarily conserved from yeast to humans. Recent studies suggest that Tom1, Tom1L1 and Tom1L2 subfamily of VHS domain proteins, which do not exist in yeast, are emerging as novel regulators for post-Golgi trafficking and signaling.

A role of Rab7 in stabilizing EGFR-Her2 and in sustaining Akt survival signal.

Rab7 plays an important role in regulating endocytic traffic. In view of an emerging role of membrane traffic in signaling and diseases, we have examined the possible role of Rab7 in oncogenesis. The role of Rab7 was investigated using shRNA-mediated knockdown in A431 and MCF7 cancer cells. To our surprise, Rab7 knockdown effectively suppressed anchorage-independent growth of cancer cells in soft agar. Anoikis (matrix-detachment triggered apoptosis) was enhanced, while the level of phosphorylated (active) Akt (which is a key survival factor) was significantly reduced. Also intriguing was the observation that EGFR and Her2 levels were significantly reduced when Rab7 was knocked-down. More robust reduction of EGFR and Her2 levels was observed when knocked-down cells were treated with HSP90 inhibitor geldanamycin (GA). Low concentration of GA (50-100 nm)-induced apoptosis of the Rab7 knocked-down cells but not control cells, suggesting that Rab7 and HSP90 together contribute to the optimal stability of EGFR and Her2 as well as to protect cancer cells from apoptosis. Rab7 seems to protect EGFR and Her2 from proteosome-mediated degradation. These results suggest that Rab7 is likely involved in protecting EGFR and Her2 from being degraded by the proteosome and in maintaining optimal Akt survival signal (especially during cell detachment or when HSP90 is inhibited). Rab7 is potentially a novel target for combinatory therapy with Hsp90 inhibitors.

RILP inhibits proliferation, migration, and invasion of PC3 prostate cancer cells.

RILP (Rab-interacting lysosomal protein) is a key regulator of lysosomal transport and a potential tumor suppressor. However, the role of RILP in prostate cancer and the underlying mechanism of RILP in regulating the proliferation, migration, and invasion of prostate cancer cells remain to be studied. In this study, we confirmed RalGDS (Ral guanine nucleotide dissociation stimulator) as the interaction partner of RILP in PC3 prostate cancer cells. Immunofluorescence microscopy showed that RILP recruits RalGDS to the lysosomal compartment. We found that RILP inhibits the activation of RalA and downstream effector RalBP1, and negatively regulates the downstream molecular phosphorylation of Ras. We showed that RILP inhibits the proliferation, migration, and invasion of PC3 prostate cancer cells, which may give rise to novel ideas for cancer treatment.

Rab36 regulates the spatial distribution of late endosomes and lysosomes through a similar mechanism to Rab34.

Small GTPase Rab36 is homologous to Rab34 with 56% amino acid sequence identity. Rab34 was characterized as a Golgi-associated Rab protein and regulates lysosomal positioning through interaction with RILP; however, the properties and functions of Rab36 have not been investigated. To investigate Rab36, we constructed EGFP-Rab36 wild type, the active GTP-bound mutant EGFP-Rab36Q116L and negative GDP-bound mutant EGFP-Rab36T71N. Expression of EGFP-Rab36 wild type revealed that Rab36 co-localized with Golgi markers GM130, Syntaxin 5 and TGN46 in Hela cells, indicating Rab36 is associated with Golgi apparatus. Over-expression of Rab36 induced late endosome and lysosome clustering around the Golgi apparatus, marked by LBPA, CD63, Lamp1 and Lamp2, without effects on early endosomal compartment marked by EEA1. GST-pulldown assay demonstrated that Rab36 can also interact with RILP. In addition, the binding region for Rab36 is in the C-terminal region (aa199-401) of RILP. Our data suggested that Rab36 may regulate the spatial distribution of late endosomes and lysosomes through a similar mechanism to Rab34.

Rab26 suppresses migration and invasion of breast cancer cells through mediating autophagic degradation of phosphorylated Src.

Rab proteins play crucial roles in membrane trafficking. Some Rab proteins are implicated in cancer development through regulating protein sorting or degradation. In this study, we found that the expression of Rab26 is suppressed in the aggressive breast cancer cells as compared to the levels in non-invasive breast cancer cells. Over-expression of Rab26 inhibits cell migration and invasion, while Rab26 knockdown significantly promotes the migration and invasion of breast cancer cells. Rab26 reduces focal adhesion association of Src kinase and induces endosomal translocation of Src. Further experiments revealed that Rab26 mediates the autophagic degradation of phosphorylated Src through interacting with ATG16L1, consequently, resulting in the suppression of the migration and invasion ability of breast cancer cells.

RILP Restricts Insulin Secretion Through Mediating Lysosomal Degradation of Proinsulin.

Insulin secretion is tightly regulated by membrane trafficking. RILP (Rab7 interacting lysosomal protein) regulates the endocytic trafficking, but its role in insulin secretion has not been investigated. In this study, we found that overexpression of RILP inhibited insulin secretion in both the ß-cell lines and freshly isolated islets. Consequently, the expression of RILP in islets suppressed the ability to recover the glucose homeostasis in type 1 diabetes mice upon transplantation. Of physiological relevance is that RILP expression was upregulated in the diabetic mouse islets. Mechanistically, overexpression of RILP induced insulin granule clustering, decreased the number of proinsulin-containing granules in ß-cells, and significantly promoted proinsulin degradation. Conversely, RILP depletion sustained proinsulin and increased insulin secretion. The proinsulin degradation induced by RILP expression was inhibited by lysosomal inhibitors and was Rab7-dependent. Finally, we showed that RILP interacts with insulin granule-associated Rab26 to restrict insulin secretion. This study presents a new pathway regulating insulin secretion and mechanically demonstrates a novel function of RILP in modulating insulin secretion through mediating the lysosomal degradation of proinsulin.

Rab7: roles in membrane trafficking and disease.

The endocytosis pathway controls multiple cellular and physiological events. The lysosome is the destination of newly synthesized lysosomal hydrolytic enzymes. Internalized molecules or particles are delivered to the lysosome for degradation through sequential transport along the endocytic pathway. The endocytic pathway is also emerging as a signalling platform, in addition to the well-known role of the plasma membrane for signalling. Rab7 is a late endosome-/lysosome-associated small GTPase, perhaps the only lysosomal Rab protein identified to date. Rab7 plays critical roles in the endocytic processes. Through interaction with its partners (including upstream regulators and downstream effectors), Rab7 participates in multiple regulation mechanisms in endosomal sorting, biogenesis of lysosome [or LRO (lysosome-related organelle)] and phagocytosis. These processes are closely related to substrates degradation, antigen presentation, cell signalling, cell survival and microbial pathogen infection. Consistently, mutations or dysfunctions of Rab7 result in traffic disorders, which cause various diseases, such as neuropathy, cancer and lipid metabolism disease. Rab7 also plays important roles in microbial pathogen infection and survival, as well as in participating in the life cycle of viruses. Here, we give a brief review on the central role of Rab7 in endosomal traffic and summarize the studies focusing on the participation of Rab7 in disease pathogenesis. The underlying mechanism governed by Rab7 and its partners will also be discussed.

Correction: A RasGAP, DAB2IP, regulates lipid droplet homeostasis by serving as GAP toward RAB40C.

[This corrects the article DOI: 10.18632/oncotarget.19960.].

Rab34 regulates adhesion, migration, and invasion of breast cancer cells.

The small GTPase Rab34 regulates spatial distribution of the lysosomes, secretion, and macropinocytosis. In this study, we found that Rab34 is over-expressed in aggressive breast cancer cells, implying a potential role of Rab34 in breast cancer. Silencing Rab34 by shRNA inhibits cell migration, invasion, and adhesion of breast cancer cells. Rab34 specifically binds to the cytoplasmic tail of integrin ß3, and depletion of Rab34 promotes the degradation of integrin ß3. Interestingly, EGF induces the translocation of Rab34 to the membrane ruffle, which is greatly enhanced by the expression of Src kinase. Accordingly, Rab34 is tyrosine phosphorylated by Src at Y247 residue. A mutant mimicking phosphorylated form of Rab34 (Rab34Y247D) promotes cell migration and invasion. Importantly, the tyrosine phosphorylation of Rab34 is inhibited in cells in suspension, and increased with the cells re-adhesion. In addition, Rab34Y247D promotes cell adhesion, and enhances integrin ß3 endocytosis and recycling. The results uncover a role of Rab34 in migration and invasion of breast cancer cells and its involvement in cancer metastasis, and provide a novel mechanism of tyrosine phosphorylation of Rab34 in regulating cell migration, invasion, and adhesion through modulating the endocytosis, stability, and recycling of integrin ß3.

Interorganellar regulation of lysosome positioning by the Golgi apparatus through Rab34 interaction with Rab-interacting lysosomal protein.

We present evidence to suggest the existence of a regulatory pathway for the Golgi apparatus to modulate the spatial positioning of otherwise distantly located lysosomes. Rab34, a new member of the Rab GTPase family, is associated primarily with the Golgi apparatus. Expression of wild-type or GTP-restricted but not GDP-restricted versions of Rab34 causes spatial redistribution of lysosomes from the periphery to the peri-Golgi region. The regulation of lysosomal positioning by Rab34 depends on its association with the membrane mediated by prenylation and its direct interaction with Rab-interacting lysosomal protein (RILP). This biological activity, mediated by Rab34-RILP interaction, is dependent on Lys82 in the switch I region. Our results have uncovered a novel mechanism for the Golgi apparatus to regulate the spatial distribution of another organelle.

A unique region of RILP distinguishes it from its related proteins in its regulation of lysosomal morphology and interaction with Rab7 and Rab34.

Rab7 and Rab34 are implicated in regulation of lysosomal morphology and they share a common effector referred to as the RILP (Rab-interacting lysosomal protein). Two novel proteins related to RILP were identified and are tentatively referred to as RLP1 and RLP2 (for RILP-like protein 1 and 2, respectively). Overexpression of RILP caused enlarged lysosomes that are positioned more centrally in the cell. However, the morphology and distribution of lysosomes were not affected by overexpression of either RLP1 or RLP2. The molecular basis for the effect of RILP on lysosomes was investigated, leading to the demonstration that a 62-residue region (amino acids 272-333) of RILP is necessary for RILP's role in regulating lysosomal morphology. Remarkably, transferring this 62-residue region unique to RILP into corresponding sites in RLP1 rendered the chimeric protein capable of regulating lysosome morphology. A correlation between the interaction with GTP-bound form of both Rab proteins and the capability of regulating lysosomes was established. These results define a unique region in RILP responsible for its specific role in regulating lysosomal morphology as well as in its interaction with Rab7 and Rab34.

Tyrosine phosphorylation of Rab7 by Src kinase.

The small molecular weight GTPase Rab7 is a key regulator for late endosomal/lysosomal membrane trafficking, it was known that Rab7 is phosphorylated, but the corresponding kinase and the functional regulation of Rab7 phosphorylation remain unclear. We provide evidence here that Rab7 is a substrate of Src kinase, and is tyrosine-phosphorylated by Src, withY183 residue of Rab7 being the optimal phosphorylation site for Src. Further investigations demonstrated that the tyrosine phosphorylation of Rab7 depends on the guanine nucleotide binding activity of Rab7 and the activity of Src kinase. The tyrosine phosphorylation of Rab7 is physiologically induced by EGF, and impairs the interaction of Rab7 with RILP, consequently inhibiting EGFR degradation and sustaining Akt signaling. These results suggest that the tyrosine phosphorylation of Rab7 may be involved in coordinating membrane trafficking and cell signaling.